Medical Biology - Theses

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Author: Preston, Simon Peter × End date: 2019 × Start date: 2011 × Type: PhD thesis × Subject: apoptosis × Subject: necroptosis ×

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    Defining programs of cell death that can be harnessed to impact on outcomes of chronic viral infection
    Preston, Simon Peter ( 2019)
    Pathogens causing chronic infections have successfully evolved mechanisms to subvert host immunity. Excessive and inappropriate inflammation together with attrition of repeatedly overstimulated high affinity T cells leads to abrogated immunity and persistence of pathogens such as HIV and HBV. T cell exhaustion has been touted as a prelude to T cell deletion during these infections, however, studies indicate that high affinity T cell clones are deleted at the onset of infection. The T cells that remain have lower affinity for pathogen epitopes and hence their response is weaker and more easily antagonised by inhibitory networks, including T-regulatory (Treg) cells. The killing of immune effector cells during chronic overwhelming infections is juxtaposed to the pathogen’s attempts to promote survival of infected target cells. Keeping infected cells alive is imperative for the maintenance of a microbial replicative niche. In this body of work, I dissected the role of host cell molecules and how they contribute to the death and survival of immune and infected cells. Necroptosis did not contribute to the loss of highly functional virus-specific CD8+ T cells during the course of infection. In contrast, when I interfered with death receptor signalling there was a modest rescue of functional CD8+ T cells. This gain in immune function, however, did not translate to improved viral control. The same mechanism I used to promote the survival of T cells made infected target cells refractory to death receptor mediated killing and therefore, offset any gain in immune function. Whilst examining the role of necroptosis in chronic infection, I made the discovery that the necroptotic inducer molecule, RIPK3, has additional non-necroptotic roles. Ripk3-/- mice cleared LCMV with enhanced kinetics compared to wild-type mice and mice that lacked the necroptotic executioner MLKL. I found that in the absence of RIPK3, chronically infected mice had impaired IFNβ responses. Excessive and prolonged IFNβ production is known to impair immunity. This may partially explain why mice lacking RIPK3 had enhanced numbers of granzyme B expressing T cells and controlled infection better than WT animals. The host-viral dynamics that favour displacement of highly functional cells with poorly activated cells makes the immune system highly vulnerable to inhibition through the activity of Treg cells. I next investigated the role of Treg cells in immune dysfunction during chronic infections and I was particularly interested in the cell death and cell survival pathways that contributed to the turnover and accumulation of these cells. I utilised mice with a Treg-specific deletion of Casp8. These mice had twice as many Treg cells as wild-type mice at steady state. Surprisingly, when these mice were infected with chronic LCMV, only 25% of the animals survived to 145 days post infection. Moribund animals succumbed to overt T cell activation and autoimmunity due to a precipitous drop in Treg cell numbers. Survivors, intriguingly, eliminated LCMV in most organs consistent with a massive gain in immune function. The death of the Treg cells was due to necroptosis. When I ablated the necroptotic pathway, through the deletion of Mlkl, I completely prevented the loss of Treg cells and the fatal immune pathology in Treg conditional caspase-8 deficient mice. I found that differential expression of RIPK3 and MLKL in Treg cells made them highly susceptible to necroptosis during chronic infection compared to Tconv cells. This was also the case for human Tregs and I was able to preferentially kill these cells, over Tconv cells, in vitro by driving necroptosis with a clinical stage caspase-8 antagonist called emricasan. Necroptosis is a lytic form of cell death that promotes inflammation and it has been implicated in chronic liver disease. I initially investigated if necroptosis in the liver contributed to the control of chronic LCMV, HBV or the malaria parasite Plasmodium berghei. Ablation of necroptosis had no impact on liver-pathogen dynamics and no impact on general liver function and architecture. In many cell types caspase-8 inhibits death receptor induced necroptosis. So, I reasoned that this molecule must be inhibiting induction of necroptosis in the liver of infected animals. I examined this by infecting mice that had a conditional loss of caspase-8 within hepatocytes. Despite abundant, infection driven, death ligands I observed no necroptosis in the liver. Even drug induced ablation of NF-ĸb survival signalling, downstream of TNF, failed to promote liver necroptosis in the aforementioned scenarios. The liver’s inability to undergo necroptosis was confirmed in mice with a human chimeric liver. I showed this refractoriness was due to liver repression of RIPK3 in humans and mice. The work conducted in this thesis provides important insights into the cell death pathways that are engaged in diverse cell types during chronic viral infections and I provide evidence that antagonising them therapeutically may lead to better clinical outcomes.